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Simple and Fast Rolling Circle Amplification-Based Detection of Topoisomerase 1 Activity in Crude Biological Samples

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Rapid screening for human-pathogenic Mucorales using rolling circle amplification.

S Dolatabadi¹, M J Najafzadeh, G S de Hoog

¹CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands; Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, the Netherlands.

|September 5, 2014

Summary

A new DNA amplification method, rolling circle amplification (RCA), rapidly identifies common mucormycosis species. This sensitive and specific technique aids in early diagnosis and contamination source tracking for better patient outcomes.

Keywords:

Mucorales environmental screening mucormycosis rolling circle amplification

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Area of Science:

Mycology
Molecular Biology
Medical Diagnostics

Background:

Mucormycosis is a severe fungal infection often seen in immunocompromised patients, particularly those undergoing allogeneic stem cell transplantation.
Environmental transmission is common, highlighting the need for rapid identification of both the pathogen and its source.
Early and accurate diagnosis is critical for effective treatment and improved patient prognosis.

Purpose of the Study:

To introduce rolling circle amplification (RCA) as a novel method for the rapid molecular identification of key mucormycosis-causing fungal species.
To evaluate the sensitivity, specificity, and reproducibility of RCA for detecting virulent fungal pathogens.
To assess the suitability of RCA for large-scale screening and early detection of mucormycosis.

Main Methods:

DNA extraction from target fungal species.
Utilizing rolling circle amplification (RCA), an isothermal DNA amplification technique.
Testing for amplification of DNA from six virulent mucormycosis species: Rhizopus microsporus, R. arrhizus var. arrhizus, R. arrhizus var. delemar, Mucor irregularis, Mucor circinelloides, and Lichtheimia species.

Main Results:

Successful DNA amplification was achieved for all targeted virulent mucormycosis species using RCA.
The RCA method demonstrated high specificity, with no cross-reactivity observed between the different fungal species.
The technique proved to be sensitive and reproducible, indicating its reliability for molecular identification.

Conclusions:

Rolling circle amplification (RCA) is a highly sensitive, specific, and reproducible isothermal DNA amplification technique.
RCA enables the rapid molecular identification of common and virulent mucormycosis pathogens.
This method is suitable for early diagnosis and large-scale screening, aiding in the control of mucormycosis outbreaks.